Flow cytometry is used to quantitatively measure physical or chemical characteristics of particles in liquid samples as they are presented into a focused light beam. Typically, this is achieved by focusing the light beam, generated by a Mercury-Vapour lamp or a laser, onto a continuous stream of liquid, known as sheath liquid, which contains samples of particles that are injected into the sheath liquid as a narrow laminar stream. The sheath liquid passes through a flow cell where it is channelled into a fine stream normally between 50 and 250 μm in diameter. The light beam is either focused onto the stream whilst it is within a quartz region of the flow cell, or once the stream has left the flow cell. This is known as either “in quartz” analysis or “in air” analysis, respectively. The point where the light/laser strikes the stream is known as the interaction region.
As the particles pass through the flow cell they enter the light beam and scatter light. Collection optics direct the light scattered by the particles and the fluorescence light emitted by the particles into a number of light detectors. All of these light scatter and fluorescence measurements can be performed simultaneously on a single particle. The data for each parameter is normally sent in real time from the flow cytometer to a computer.
The flow rate of liquid through the focused beam is usually around 10 ms−1 which allows a maximum of approximately 2,000 to 10,000 cells or particles to be analysed per second.
In addition to flow cytometry analysis described above, flow cytometry can be used to physically sort cells using information from the various detectors as discriminators. Sorting enables purification of a particular cell type from a mixture. The standard method of sorting cells by flow cytometry is known as droplet deflection sorting. It relies on the use of a piezoelectric transducer in the flow cell to create droplets of sheath liquid. An alternating electrical current is passed across the transducer causing the flow cell to vibrate up and down at the same frequency as the current. The vibration of the flow cell causes undulations to form in the sheath liquid once it has left the flow cell. Further downstream from the flow cell the undulations in the stream of sheath liquid become more and more defined until the stream breaks up into droplets. The last undulation in the stream before the stream breaks up into droplets is known as the last attached droplet.
If a particle is to be collected, then an electric charge is placed on the sheath liquid at the exact time the particle is in the last attached droplet. The charge occurs for the duration of one vibration of the piezoelectric crystal in the transducer. This results in a single droplet, containing the one particle to be sorted, being charged. Further downstream from the flow cell the stream of droplets passes between two plates, one positively and one negatively charged. As the charged droplet passes between the plates it is diverted from the main stream of droplets enabling it to be collected. This sorting process can be performed at a rate of several thousands times per second using a modern cytometer.
Flow cytometers that use droplet deflection sorting, however, are sophisticated instruments that require at least daily alignment by a highly skilled operator. Setting up the sorting is also difficult and requires a number of calibrations including calculation of the length of time that it takes a particle to travel from the interaction region to the last attached droplet. This length of time is known as the droplet delay. Once the sorting has been set up it has to be monitored closely to ensure that the droplet delay does not change.
A limitation of droplet deflection sorting is that it is not able to create droplets that contain more than one particle. A further problem with droplet deflection sorting is that it can create aerosols and is therefore not suitable for the analysis of biologically hazardous samples. The difficulties with the use of droplet delay sorting have restricted the use of the technology to specialised research laboratories.
An alternative form of flow cytometry sorting is described in U.S. Pat. No. 5,030,002 “Method and apparatus for sorting particles with a moving capture tube” (incorporated herein by reference). This sorting process uses a capture tube that is mechanically moved in and out of the sample stream to capture a particle. The cytometer is formed from a housing having an upper portion and a lower portion, which is shaped to form a nozzle. The lower portion of the housing is coupled to a container containing a sample liquid to be analysed. In use, the sample liquid is pumped into the housing via a tube using a pump. In addition, the housing is connected to a source of sheathing liquid, which is pumped into the housing along a tube using a pump. The upper part of the housing is connected via a tube to a waste container for catching waste sheathing liquid. The upper part of the housing is adapted to accept a capture tube. The capture tube is movable between first and second positions using an actuator, such as a solenoid or the like. The capture tube is coupled via a silicon hose to a container that collects the produced sample in a stream of liquid. In order to be able to analyse the sampled liquid, the cytometer includes a detection system formed generally from a radiation source such as a laser and one or more detectors. The detection system is adapted to detect properties of particles flowing through an interaction region. In use, the system operates to analyse individual particles contained within the sample liquid. This is achieved by having the sample liquid pumped up the tube into the housing. Simultaneously, sheathing liquid is pumped to the housing via the tube. The sheathing liquid is pumped into the housing under pressure so as to generate a focused stream of liquid surrounding the sampling liquid. As the sheathing liquid and the sampling liquid are forced through the nozzle, this causes the sampling liquid to form a thin stream containing a laminar stream of individual particles from the sample. As the sample particles pass through the interaction region, the detection system operates to analyse one or more properties of each cell or particle. A signal is then transferred via a control system to the actuator control the position of the capture tube. In use, when the capture tube is in a first position, particles contained in the laminar stream are directed through the capture tube to a collection container. When the capture tube is in a second position, the laminar stream containing particles are directed into the waste container. Accordingly, this allows the system to sort particles by detecting certain properties of the particles and then directing the particles either into a collection container or into the waste container.
In many cases, however, it is desirable to able to produce predetermined quantities and in particular predetermined concentration particles in a sample and this generally not possible with this arrangement. In particular, even when the actuator is placed in the second position, sheathing liquid will still be transmitted through the capture tube and the hose into the collection container. Accordingly, it is very difficult to direct a predetermined number of particles into the collection container and maintain a desired concentration of particles.
For example, this sorting process is utilised by the Becton Dickinson FACScalibur™ flow cytometer. This flow cytometer is simple to operate and requires no calibration or complex set-up procedure to be performed prior to sorting particles. It is simply a case of switching on the instrument, analysing a sample and sorting the particles of interest into a relatively large collection volume.
The FACScalibur™ flow cytometer has a length of silicon tubing (sort line) connected to the capture tube at the top of the flow cell. Particles sorted by the capture tube travel down this sort line into the desired type of collection vessel. As described above, there is a constant stream of sheath liquid travelling through the sort line that carries the sorted particles into the collection vessel. This constant stream of sheath liquid has to be collected along with the sorted particles resulting in the sorted particles being contained in a large volume of liquid (typically 1 to 50 ml, depending on the time of the collection process). The particles therefore have to be concentrated to much smaller volumes before they can be analysed or visualised with a microscope.
Furthermore, there are a number of applications of flow cytometry cell sorting that require sorted particles to be in a small volume of liquid. For example, the preparation of quality control samples of particles that contain an exact number of particles in a small volume of liquid. In this case, whilst a predetermined number of particles can be directed into the sort line, the particles will tend to travel along the line at different speeds because flow within the line is not laminar. The flow is slower close the wall of the line due to friction between the water molecules and the line wall. As a result, the particles become dispersed throughout the sheathing liquid and it is therefore not possible to control the concentration of the particles received at the container.
U.S. Pat. No. 5,030,002 also describes the use of a filter connected to the sort line to capture and concentrate the sorted particles. However, even using these techniques, the number of particles contained in a sample volume cannot be suitably controlled.
Accordingly, the FACScalibur™ flow cytometer cannot be used to produce droplets that contain exact numbers of particles. This is because the silicon sort line must be connected to the flow cell to carry the sorted particles away from the flow cell to a position where they can be collected. When sorted particles travel through the tubing they spread out and will no longer be in a single drop.
A flow cytometer that utilises droplet deflection sorting can be used to produce droplets that contain single particles. However, the droplet size that is produced by a cytometer that uses droplet deflection is typically limited to less than 400 μm in diameter, which can have significant disadvantages in many applications. For example, the preparation of freeze dried droplets that contain exact numbers of particles for quality control applications requires a freeze dried droplet that is large enough to be easily manipulated and visible to the human eye. Droplets from a droplet deflection cytometer are not large enough for this application. Furthermore, it is not possible to produce droplets with a droplet deflection cytometer that contain more than one particle.
Furthermore, droplet deflection sorting is less suitable as a production process than a flow cytometer that uses a catcher tube to sort particles because of the difficulties with calibrating and setting up the droplet deflection flow cytometer. In particular, a flow cytometer that utilises droplet deflection sorting requires a highly skilled operator and needs continuous monitoring during the sorting process.
The present inventors have developed modifications to a cytometer that enable defined numbers of particles in various small volumes of liquid to be formed and collected.